Current influenza vaccines are targeted to induce immune responses to the variable antigens hemagglutinin and neuraminidase, which are effective for strain-specific protection. However, such vaccination does not provide protection against the emergence of antigenically distinct strains as shown by the failure to control the 2009 H1N1 pandemic at the early stage of its outbreak. The goal of our proposed project is to develop novel influenza vaccines that will induce broadly cross protective immunity against antigenically drifted strains in the absence of adjuvants. To achieve this goal, novel approaches will be proposed to develop a highly conserved antigenic target in an immunogenic form and to incorporate this into the influenza vaccination. A promising candidate as a conserved antigenic target is the membrane protein M2 containing a highly conserved extracellular domain. M2 on virus-like particles (VLPs) in a membrane-anchored form (M2 VLPs) will be in a conformation enabling M2 to be immunogenic and confer broadly cross-protective M2 immunity even without an adjuvant. We hypothesize that influenza vaccines containing highly conserved antigenic targets such as influenza M2 VLPs will induce broadly cross-protective and heterosubtypic immunity. To test this hypothesis, in specific aim 1, recombinant VLP vaccines containing novel constructs of the tetrameric M2 extracellular domain in a membrane-anchored chimeric form will be generated and their cross protective efficacy will be evaluated in comparison with the wild type M2 protein. We will also propose a novel approach to overcome the limitation of strain-specific protection by current vaccines and weak cross-protective immunity to M2. Specific aim 2 will investigate action mechanisms by which conserved M2 based immunity enhances the breadth of cross protection. Also, immune correlates contributing to cross protection will be determined using traditional and novel approaches including depletion of specific immune components. In specific aim 3, the cross-protective efficacy of promising vaccine candidates and the duration of cross protection will be further evaluated in ferrets, which is a more relevant animal model for testing pre-clinical vaccines for humans. Improving the breadth of cross protective immunity against influenza viruses after vaccination without using adjuvant is a desirable and practical approach applicable to humans and critically important for advancing the vaccine field.